1. Field of the Invention
The present invention relates to the field of electrophysiology, more specifically to the field of patch clamp electrophysiology, and most specifically to automated patch clamp electrophysiology instruments and systems, as well as methods for their use.
Electrophysiology includes, but is not limited to, the electrical properties of biological membranes, such as transmembrane potential(s) and the transmembrane flow of current through membrane-associated ion channels.
2. Description of Related Art
The basic concepts of patch clamp electrophysiology are known in the art. For reference, see Single Channel Recording, by Sakmann and Neher, all editions to date, The Axon Guide by Axon Instruments, Inc., and Ion Channels of Excitable Membranes by B. Hille, 3rd edition, all of which are incorporated herein by reference in their entirety for all purposes.
Electrophysiology may be studied using a variety of techniques. See, e.g., the above-mentioned The Axon Guide, Single Channel Recording, and Ion Channels of Excitable Membranes, all of which are incorporated herein by this reference. For example, the patch clamp technique may be used to study the electrical properties of membranes, including the flow of current through ion channels in the membranes. Typical patch clamp recording configurations are whole cell in single channel mode, both modes are known in the field. Typical whole cell recording requires gaining access to the cell interior usually facilitated by suction, zapping and antibiotic. FIG. 1 shows a fundamental equivalent electrical circuit, which models aspects of membrane electrical behavior. Here, the cell resistance R, also is known as the membrane resistance or the leak resistance. The pipette resistance Rs often is referred to and is usually indistinguishable from the access, resistance or the series resistance.
Automated patch clamp devices to date voltage clamp a single cell per chamber, whether that chamber is a well in a cartridge, a flow through channel, or the inside or outside of a pipette. This may limit the throughput, limit the applications, and set a lower floor to the cost per data point, for reasons that will be described. Applicant also makes no representation that the following discussion is material to patentability, and the following discussion shall not be construed as an admission against interest in any manner.
Generally, there are two approaches used in today's automated patch clamps. These two approaches differ primarily in the methodology used to manage fluid exchange and to perform electrophysiological recording.
Approach I: Multiple Add, Reads, and Washes.
This approach is characterized by a single cell per well with fluidics capable of repetitively adding and removing fluids from each well independently. This approach utilizes fluidics to wash cells with buffer and add multiple compounds so that multiple data points can be acquired from each cell.
Typical devices are the PatchXpress® system from Molecular Devices Corporation, the Q-Patch from Sophion, and the Port-A-Patch from Nanion. These kinds of devices may be very flexible and can perform a wide variety of experiments, but their throughput is low. For example, the sixteen channel PatchXpress® 7000A can collect a maximum of about 240 data points per day (i.e., four data points per well, eight wells per SealChip® cartridge, and eight SealChip® cartridges per day, assuming 50% success rate on each SealChip cartridge, e.g., 50% of 16 wells per SealChip® cartridge=8 wells, experiment duration of 30-60 minutes).
Such systems can determine control responses on the same cell to which the test compound is applied, whether the applied compound modifies a voltage gated ion current (VGIC) or a ligand gated ion current (LGIC). By testing the viability of the cells and taking advantage of random access fluidics it is largely possible to guarantee that all compounds are tested, and other than statistical robustness there is no need to test a compound on more than one cell.
Approach II: Add and Read.
In an “add and read” system, no provision is made to wash the chamber with buffer or to replace or typically even top up the compounds; thus, only a single data point is acquired from each chamber.
This approach is embodied in the IonWorks® HT system from Molecular Devices Corporation. This system is limited to addition of solution and reading of the response (add and read). Compounds are added in groups, irrespective of the viability of the cells in the wells. The advantage of this system is that it is much simpler than a system that allows fluid exchange. The disadvantage is that since the typical rate of forming a successful whole cell recording configuration is in the vicinity of 60% to 70%, compounds have to be applied redundantly to guarantee that there is at least one successful recording of each compound. Usually the redundancy is fourfold, but eightfold or higher redundancy is required when the cells form the whole cell configurations at lower success rates. This redundancy both increases the cost per data point and decreases the throughput.
Like any measurement system, the add-and-read system can only work if a control measurement can be made to which the test compound response will be compared. In the case of VGICs, this is easy. First, the response to a series of voltage commands is measured before adding the test compound. This is the control response, representing 100%. Second, the response to the same series of voltage commands is measured after the test compound is added. Dividing this by the control response provides the percentage inhibition or activation.
It is not possible to make the control measurement in the case of LGICs because it would be necessary to add an agonist to determine the control response, then wash it out before adding the test compound (typically a mixture of test compound and agonist). This cannot be done in a conventional add-and-read system. One possible solution would be to measure the control response in one cell in a first well and add the test compound to a different cell in a second well. However, the variability in expression of ion channels in any given cell can range from zero to several times the average, thus the response to control compounds will have similar variability. The resulting errors involved in using a single cell as a control and a different cell for testing are unacceptable.
The throughput of add-and-read systems is high, relative to approach I. For example, in a typical 8-hour day, six to ten 384 well PatchPlate™ substrates will be processed, since the typical duration of an experiment is about 45-75 minutes. With the usual four-fold redundancy, 96 compounds will be delivered per 384 chamber PatchPlate™ substrate. Thus, the number of data points per day is nominally 600-1000.
For dose response applications in an add-and-read system, dose response curves are obtained by applying compounds at different doses to different groups of four chambers (assuming four-fold redundancy).
For either Approach I or Approach II, one of several compound delivery approaches can be utilized.
Fluidics I: Multiple Tips, Non-Random Access
In this approach, a multi-tip head is used to pick up many different compounds at once, to enhance or maximize throughput. In a basic implementation there is a one to one mapping between compound locations and dispense locations. Thus, if the cell at a particular patching location is unviable the compound will be dispensed at that location regardless. To guarantee that all compounds are tested, the compounds must be applied redundantly to multiple chambers, typically four or more chambers.
Fluidics II: Single Tip, Random Access
In this approach, a single-tip fluidics dispenser adds a compound to one chamber at a time. For example, the PatchXpress® system uses a Cavro® (Tecan) dispenser for this purpose. The advantage of this approach is that the software can choose any compound from any position. By analogy with computer memory this selection flexibility is known as “random access”. The advantage of random access is that compounds do not have to be used in any particular order and all compounds can be accessed and applied irrespective of the success of the patching process in any given patch clamp chamber. The disadvantage of this approach is speed. There is simply a limit as to how fast a single pipetting head can pick up a disposable tip, aspirate compound, and dispense it in a chamber. A full cycle may take about fifteen seconds.
Fluidics III: Multiple Tips, Random Access
A modification of the Fluidics II approach is to use multiple tips with random access. This can be achieved through the use of multiple independent tip robots that are controlled through a central software application. Alternatively, approaches are currently available whereby the flow of solution in a multichannel tip head can be controlled for each single tip. Additionally, robots exist that allow the control of inter-tip spacing in a multi-tip head, thus allowing for spatially and temporally controlled access to multiple wells from a multi-tip head.
In addition to commercial automated electrophysiology products currently available from companies such as Molecular Devices, Sophion, Nanion, etc., there are a number of patents and patent applications related to automated electrophysiology. Examples include U.S. patent application Ser. No. 09/900,627 (i.e., U.S. Patent Application Publication No. US 2002/0053915 A1), assigned to Bristol-Myers Squibb Company, and International Patent Publication No. WO 99/66329, assigned to CeNeS.
The existing devices and the existing literature indicate that there is a need in the field for a device and methods to automatically and accurately record electrophysiological signals with high throughput and high resolution for a variety of applications in a cost effective manner.
Other limitations of prior patch clamp techniques exist. During patch clamp recording, users may enable a variety of circuit compensation mechanisms, some of which may be found on commercial patch clamp amplifiers, including the Axopatch series of amplifiers by Molecular Devices Corporation. For example, users may nullify the electrode and/or whole cell capacitance, and activate series resistance compensation. Users also may enable passive leak subtraction.
During electrophysiological recording, it often is advantageous to estimate the electrical properties of the cell or preparation being analyzed. For example, users may want to estimate the whole cell capacitance or determine if the series resistance has changed during the course of the experiment. One approach that may be used is to apply a square voltage pulse to the cell being patch clamped, and then to analyze the current response of the cell. The current response to a square wave is a low-pass filtered, single- or multi-exponential decay, where the time constants of the waveform are a function of the resistance and capacitance of the cell and the instrumentation. The “Membrane Test”function in the industry standard software package pClamp (Molecular Devices Corporation) uses this approach to estimate various experimental parameters. See, e.g., pCLAMP 9—DATA ACQUISITION AND ANALYSIS FOR COMPREHENSIVE ELECTROPHYSIOLOGY—User's Guide, Axon Instruments, Inc. (August 2003). FIG. 2B shows an example of the data collected and estimated from the Membrane Test protocol in pClamp.
Unfortunately, in approaches such as that employed in Membrane Test, it may be very difficult accurately to curve fit the current response after electronic compensation has been enabled in the amplifier. For example, if whole-cell membrane capacitance compensation has been enabled, the passive membrane current response to a square voltage pulse will be significantly smaller or even zero, and thus accurate curve-fitting will be difficult or impossible, due to a lower signal-to-noise ratio.
Thus, there is a need for a system for estimating electrophysiological parameters during patch clamp recording, when compensation for at least some of these parameters has been enabled in the amplifier.